Gene Expression Profiling Defines ATP as a Key Regulator of Human Dendritic Cell Functions This information is current as Nathalie Bles, Michael Horckmans, Anne Lefort, Frédéric of September 30, 2021. Libert, Pascale Macours, Hakim El Housni, Frédéric Marteau, Jean-Marie Boeynaems and Didier Communi J Immunol 2007; 179:3550-3558; ; doi: 10.4049/jimmunol.179.6.3550 http://www.jimmunol.org/content/179/6/3550 Downloaded from Supplementary http://www.jimmunol.org/content/suppl/2008/03/12/179.6.3550.DC1 Material http://www.jimmunol.org/ References This article cites 36 articles, 19 of which you can access for free at: http://www.jimmunol.org/content/179/6/3550.full#ref-list-1 Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision by guest on September 30, 2021 • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2007 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology Gene Expression Profiling Defines ATP as a Key Regulator of Human Dendritic Cell Functions1 Nathalie Bles,2* Michael Horckmans,2* Anne Lefort,* Fre´de´ric Libert,* Pascale Macours,† Hakim El Housni,‡ Fre´de´ric Marteau,* Jean-Marie Boeynaems,*† and Didier Communi3* Extracellular ATP and PGE2 are two cAMP-elevating agents inducing semimaturation of human monocyte-derived dendritic cells (MoDCs). We have extensively compared the gene expression profiles induced by adenosine 5؅-O-(3-thiotriphosphate) (ATP␥S) ␥ and PGE2 in human MoDCs using microarray technology. At6hofstimulation, ATP S initiated an impressive expression profile compared with that of PGE2 (1125 genes compared with 133 genes, respectively) but after 24 h the number of genes regulated by ␥ ATP SorPGE2 was more comparable. Many target genes involved in inflammation have been identified and validated by ␥ quantitative RT-PCR experiments. We have then focused on novel ATP S and PGE2 target genes in MoDCs including CSF-1, MCP-4/CCL13 chemokine, vascular endothelial growth factor-A, and neuropilin-1. ATP␥S strongly down-regulated CSF-1 re- Downloaded from ceptor mRNA and CSF-1 secretion, which are involved in monocyte and dendritic cell (DC) differentiation. Additionally, ATP␥S down-regulated several chemokines involved in monocyte and DC migration including CCL2/MCP-1, CCL3/MIP-1␣, CCL4/ MIP-1␤, CCL8/MCP-2, and CCL13/MCP-4. Interestingly, vascular endothelial growth factor A, a major angiogenic factor dis- ␥ playing immunosuppressive properties, was secreted by MoDCs in response to ATP S, ATP, or PGE2, alone or in synergy with ␥ LPS. Finally, flow cytometry experiments have demonstrated that ATP S, ATP, and PGE2 down-regulate neuropilin-1, a receptor playing inter alia an important role in the activation of T lymphocytes by DCs. Our data give an extensive overview of the genes http://www.jimmunol.org/ ␥ regulated by ATP S and PGE2 in MoDCs and an important insight into the therapeutic potential of ATP- and PGE2-treated human DCs. The Journal of Immunology, 2007, 179: 3550–3558. aturation of dendritic cells (DCs)4 in response to LPS, regulation of costimulatory molecules (2–4) and regulates various CD40 ligand, or proinflammatory cytokines is reflected chemokines and chemokine receptors (5, 6). ATP also regulates the M by a loss of endocytosis, the surface expression of stable action of LPS and other maturating agents on human DCs by inhib- MHC-peptide complexes and costimulatory molecules (CD80, iting the production of proinflammatory cytokines such as IL-12, IL- CD86), the production of cytokines like IL-12, and a shift in the ex- 1␤, TNF-␣, and IL-6 and by potentiating anti-inflammatory IL-10 by guest on September 30, 2021 pression of chemokines and their receptors allowing DC migration to (2, 7). These features of ATP-treated DCs are compatible with semi- lymphoid organs (1). In human monocyte-derived DCs (MoDCs), mature DCs whose potential involvement in central and peripheral ATP, which is released inter alia from necrotic cells, induces the up- tolerance has been previously discussed (8–10). This profile of action of ATP is similar to that of cAMP-elevating agents such as PGE2 and is indeed associated with an increase in cAMP, presumably mediated *Institute of Interdisciplinary Research, Interdisciplinaire en Biologie Humaine et by the P2Y11 receptor (4). ATP, via inhibition of IL-12 and potenti- Mole´culaire, Universite´Libre de Bruxelles, Brussels, Belgium; †Department of Med- ation of IL-10, will thus impair the initiation of a Th1 response and ical Chemistry, Erasme Hospital, Universite´Libre de Bruxelles, Brussels, Belgium; and ‡Department of Genetics, Erasme Hospital, Universite´Libre de Bruxelles, Brus- favor a Th2 response or tolerance (2, 7). Recently, we reported the sels, Belgium critical role of ATP-mediated signal transduction in triggering two Received for publication October 31, 2006. Accepted for publication July 6, 2007. targets (thrombospondin-1 (TSP-1) and IDO) involved in T cell im- The costs of publication of this article were defrayed in part by the payment of page munosuppression, suggesting a potential role of extracellular nucleo- charges. This article must therefore be hereby marked advertisement in accordance tides in immune tolerance (11). with 18 U.S.C. Section 1734 solely to indicate this fact. In the present study, we have extensively compared the target 1 This work was supported by an Action de Recherche Concerte´e of the Communaute´ genes of adenosine 5Ј-O-(3-thiotriphosphate) (ATP␥S) and PGE Franc¸aise de Belgique, by the Belgian Programme on Interuniversity Poles of Attrac- 2 tion initiated by the Belgian State, Prime Minister’s Office, Federal Service for Sci- in human MoDCs using a combination of microarray technology, ence, Technology and Culture, by grants of the Fonds de la Recherche Scientifique quantitative RT-PCR experiments, ELISAs, and flow cytometry Me´dicale, the Fonds Emile DEFAY, and the LifeSciHealth programme of the Euro- pean Community (Grant LSHB-2003-503337). N. B., M. H., and F. M. were sup- analysis. This study is the first one to provide gene expression ␥ ported by the Fonds National de la Recherche Scientifique/Fonds pour la Recherche profiles of ATP S and PGE2 in MoDCs and gives an overview of dans l’Industrie et dans l’Agriculture, Belgium. D. C. and F. L. are Research Asso- the potential actions of ATP and PGE on human DCs. ciate of the Fonds National de la Recherche Scientifique (FNRS). 2 2 N. B. and M. H. contributed equally to the work. Materials and Methods 3 Address correspondence and reprint requests to Dr. Didier Communi, Institute of Reagents Interdisciplinary Research (IRIBHM), Universite´Libre de Bruxelles, Building C (5th floor), Campus Erasme, 808 Route de Lennik, Brussels, Belgium. E-mail address: ATP, ATP␥S, PGE , forskolin, and LPS were obtained from Sigma-Aldrich. [email protected] 2 4 Abbreviations used in this paper: DC, dendritic cell; ATP␥S, adenosine 5Ј-O-(3- Preparation of MoDCs thiotriphosphate); MoDC, monocyte-derived DC; TSP-1, thrombospondin-1; NRP-1, neuropilin-1; qRT-PCR, quantitative RT-PCR; VEGF, vascular endothelial growth PBMCs were isolated from leukocyte-enriched buffy coats of healthy volun- factor. teer donors by standard density gradient centrifugation using Lymphoprep so- lution from Nycomed. PBMCs (2.5 ϫ 108) were allowed to adhere for1hand 2 Copyright © 2007 by The American Association of Immunologists, Inc. 0022-1767/07/$2.00 30 min at 37°C at 5% CO2 in air in 75-cm cell culture flasks. Nonadherent www.jimmunol.org The Journal of Immunology 3551 ␥ cells were removed and adherent cells were cultured in 15 ml of RPMI 1640 Table I. Number of genes regulated by ATP S and/or PGE2 at 2, 6, medium supplemented with 800 U/ml GM-CSF and 500 U/ml IL-4. GM-CSF 12, or 24 h in human MoDCsa and IL-4 were also added a second time 2 days after the adhesion step. Five days after the adhesion step, the purity of each cell preparation was evaluated 2h 6h 12h 24h using flow cytometry by analyzing the expression of two markers of DCs, HLA-DR and CD1a. Moreover, the absence of monocytes, lymphocytes, and ATP␥S 534 1125 595 413 mature DCs was always checked by staining cell preparation using CD14, PGE2 42 133 117 307 CD3, and CD83 markers, respectively. For our experiments, we have only ATP␥S/PGE 38 119 90 164 used cell preparations of HLA-DRϩCD1aϩ immature DCs displaying at least 2 95% purity. Cells were then plated at 106 cells/ml in 24 multiwells in complete a Evaluated by using microarray technology. medium. Agents were then added for different periods of time. Flow cytometry analysis Cells were labeled with FITC-conjugated anti-human CD83 and PE-con- is more resistant to degradation by ectonucleotidases, was used jugated anti-human CD1a, HLA-DR, CD14, CD3, and neuropilin-1 instead of ATP to avoid additional gene regulations due to its ϫ 5 ␮ (NRP-1) Abs (BD Pharmingen). Cells (2 10 ) were incubated in 100 l degradation products such as ADP and adenosine. PGE2 displays of PBS with 0.1% sodium azide for 30 min in the dark at 4°C, washed with effects similar to those of ATP on DC maturation and is also able 1 ml of PBS, and analyzed on a Cytomics FC 500 flow cytometry system to activate the cAMP pathway in MoDCs (14, 15).